Pigment protein from Cnidopus japonicus

ABSTRACT

An object of the present invention is to provide a chromoprotein derived from  Cnidopus japonicus . The present invention provides a chromoprotein derived from  Cnidopus japonicus  having the following properties: (1) the absorption maximum wavelength is 610 nm, and fluorescence is not emitted; (2) the molar absorption coefficient is 66,700 at 610 nm; and (3) the pH sensitivity of light-absorbing property is stable at between pH 4 and pH 10.

This application is the National Stage of International ApplicationPCT/JP03/07336, filed Jun. 10, 2003.

TECHNICAL FIELD

The present invention relates to a novel chromoprotein. Morespecifically, the present invention relates to a novel chromoproteinderived from Cnidopus japonicus, and the use thereof.

BACKGROUND ART

Green fluorescent protein (GFP) derived from Aequorea Victoria, ajellyfish, has many purposes in biological systems. Recently, variousGFP mutants have been produced based on the random mutagenesis andsemi-rational mutagenesis, wherein a color is changed, a foldingproperty is improved, luminance is enhanced, or pH sensitivity ismodified. Fluorescent proteins such as GFP are fused with other proteinsby gene recombinant technique, and monitoring of the expression andtransportation of the fusion proteins is carried out.

One of the most commonly used types of GFP mutant is Yellow fluorescentprotein (YFP). Among Aequorea-derived GFP mutants, YFP exhibits thefluorescence with the longest wavelength. The values ε and Φ of themajority of YEPs are 60,000 to 100,000 M⁻¹cm⁻¹ and 0.6 to 0.8,respectively (Tsien, R. Y. (1998). Ann. Rev. Biochem. 67, 509-544).These values are comparable to those of the general fluorescent group(fluorescein, rhodamine, etc.). Accordingly, improvement of the absoluteluminance of YFP is nearly approaching its limit.

In addition, cyan fluorescent protein (CFP) is another example of theGFP mutant. Of this type of protein, ECFP (enhanced cyan fluorescentprotein) has been known. Moreover, red fluorescent protein (RFP) hasbeen isolated from sea anemone (Discoma sp.). Of this type of protein,DasRed has been known. Thus, 4 types of fluorescent proteins, that are,green fluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, and red fluorescent protein, have successively been developed.The range of the spectrum has significantly been expanded.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel chromoproteinderived from Cnidopus japonicus.

The present inventors have conducted intensive studies directed towardsachieving the aforementioned object. They have designed suitable primersbased on information regarding the amino acid sequences of knownfluorescent proteins. Using these primers, they have succeeded in theamplification and cloning of a gene encoding a novel chromoprotein fromthe cDNA library of Cnidopus japonicus exhibiting a green color. Thepresent inventors have further analyzed the light-absorbing propertiesand pH sensitivity of the obtained chromoprotein derived from Cnidopusjaponicus. The present invention has been completed based on thesefindings.

That is to say, the present invention provides a chromoprotein derivedfrom Cnidopus japonicus having the following properties:

-   (1) the absorption maximum wavelength is 610 nm, and fluorescence is    not emitted;-   (2) the molar absorption coefficient is 66,700 at 610 nm; and-   (3) the pH sensitivity of light-absorbing property is stable at    between pH 4 and pH 10.

In another aspect, the present invention provides a chromoprotein havingeither one of the following amino acid sequences:

-   (a) the amino acid sequence shown in SEQ ID NO: 1; and-   (b) an amino acid sequence comprising a deletion, substitution    and/or addition of one or several amino acids with respect to the    amino acid sequence shown in SEQ ID NO: 1, and having    light-absorbing properties.

In another aspect, the present invention provides a chromoproteincapable of emitting fluorescence, which has an amino acid sequence,wherein, with respect to the amino acid sequence shown in SEQ ID NO: 1,alanine as an amino acid residue at position 28 is substituted byglycine, glutamic acid as an amino acid residue at position 41 issubstituted by methionine, cysteine as an amino acid residue at position145 is substituted by serine, and threonine as an amino acid residue atposition 158 is substituted by isoleucine.

In another aspect, the present invention provides:

a chromoprotein having an amino acid sequence wherein tyrosine as anamino acid residue at position 64 is substituted by leucine with respectto the amino acid sequence shown in SEQ ID NO: 1;

a chromoprotein having an amino acid sequence wherein tyrosine as anamino acid residue at position 64 is substituted by methionine withrespect to the amino acid sequence shown in SEQ ID NO: 1;

a chromoprotein having an amino acid sequence, wherein glutamic acid asan amino acid residue at position 41 is substituted by leucine, andphenylalanine as an amino acid residue at position 80 is substituted byglycine, with respect to the amino acid sequence shown in SEQ ID NO: 1;

a chromoprotein capable of emitting fluorescence, which has an aminoacid sequence wherein tyrosine as an amino acid residue at position 64is substituted by phenylalanine with respect to the amino acid sequenceshown in SEQ ID NO: 1;

a chromoprotein capable of emitting fluorescence, which has an aminoacid sequence wherein tyrosine as an amino acid residue at position 64is substituted by histidine with respect to the amino acid sequenceshown in SEQ ID NO: 1; and

a chromoprotein capable of emitting fluorescence, which has an aminoacid sequence, wherein cysteine as an amino acid residue at position 26is substituted by valine, cysteine as an amino acid residue at position143 is substituted by serine, and proline as an amino acid residue atposition 199 is substituted by leucine, with respect to the amino acidsequence shown in SEQ ID NO: 1.

In another aspect, the present invention provides DNA encoding theprotein of the present invention.

In another aspect, the present invention provides either one of thefollowing DNAs:

-   (a) DNA encoding the amino acid sequence shown in SEQ ID NO: 1; and-   (b) DNA encoding an amino acid sequence, which comprises a deletion,    substitution and/or addition of one or several amino acids with    respect to the amino acid sequence shown in SEQ ID NO: 1, and has    light-absorbing properties.

In another aspect, the present invention provides DNA having either oneof the following nucleotide sequences:

-   (a) the nucleotide sequence shown in SEQ ID NO: 2; and-   (b) a nucleotide sequence comprising a deletion, substitution and/or    addition of one or several nucleotides with respect to the    nucleotide sequence shown in SEQ ID NO: 2, and encoding a protein    having light-absorbing properties.

In another aspect, the present invention provides DNA having thenucleotide sequence shown in any one of SEQ ID NOS: 12, 14, 16, 18, 20,or 22.

In another aspect, the present invention provides a recombinant vectorhaving the DNA of the present invention.

In another aspect, the present invention provides a transformant havingthe DNA or recombinant vector of the present invention.

In another aspect, the present invention provides a fusion proteincomposed of the chromoprotein of the present invention and anotherprotein.

In another aspect, the present invention provides a method for analyzinga physiologically active substance, which is characterized in that theFRET (fluorescence resonance energy transfer) method is applied usingthe chromoprotein of the present invention as an acceptor protein.

In another aspect, the present invention provides a light-absorbingreagent kit comprising the chromoprotein, DNA, recombinant vector,transformant, or fusion protein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results obtained by measuring the absorption spectrumof the chromoprotein (KGr) derived from Cnidopus japonicus of thepresent invention. The transverse axis indicates the wavelength ofabsorbed light, and the vertical axis indicates absorbance.

FIG. 2 shows the pH sensitivity of the absorption spectrum of thechromoprotein (KGr) derived from Cnidopus japonicus of the presentinvention. The transverse axis indicates pH value, and the vertical axisindicates absorbance. 610 nm indicates the absorbance that is specificto the chromoprotein (KGr) derived from Cnidopus japonicus of thepresent invention, and 277 nm indicates the absorbance (light absorptionby aromatic amino acids) that is generally used as a quantitative amountof protein. In other words, it is shown that the amount of protein isconstant at 277 nm, and the absorbance at 610 nm that is specific to thechromoprotein (KGr) derived from Cnidopus japonicus of the presentinvention hardly changes in the range between pH 4 and pH 10.

FIG. 3 shows the fluorescence spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting A at position 28 by G, substituting E atposition 41 by M, substituting C at position 145 by S, and substitutingT at position 158 by I in the amino acid sequence of KGr). Thetransverse axis indicates wavelength, and the vertical axis indicatesfluorescence intensity. The term “em” indicates a fluorescence spectrum,and the term “ex” indicates an excitation spectrum.

FIG. 4 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting Y at position 64 by L in the amino acidsequence of KGr).

FIG. 5 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting Y at position 64 by M in the amino acidsequence of KGr).

FIG. 6 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting E at position 41 by L and substituting F atposition 80 by G in the amino acid sequence of KGr).

FIG. 7 shows the fluorescence and excitation spectrums of a mutant ofthe chromoprotein (KGr) derived from Cnidopus japonicus of the presentinvention (a mutant obtained by substituting Y at position 64 by F inthe amino acid sequence of KGr).

FIG. 8 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting Y at position 64 by F in the amino acidsequence of KGr).

FIG. 9 shows the fluorescence and excitation spectrums of a mutant ofthe chromoprotein (KGr) derived from Cnidopus japonicus of the presentinvention (a mutant obtained by substituting Y at position 64 by H inthe amino acid sequence of KGr).

FIG. 10 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting Y at position 64 by H in the amino acidsequence of KGr).

FIG. 11 shows the fluorescence and excitation spectrums of a mutant ofthe chromoprotein (KGr) derived from Cnidopus japonicus of the presentinvention (a mutant obtained by substituting C at position 26 by V,substituting C at position 143 by S, and substituting P at position 199by L in the amino acid sequence of KGr).

FIG. 12 shows the absorption spectrum of a mutant of the chromoprotein(KGr) derived from Cnidopus japonicus of the present invention (a mutantobtained by substituting C at position 26 by V, substituting C atposition 143 by S, and substituting P at position 199 by L in the aminoacid sequence of KGr).

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described in detailbelow.

(1) Chromoprotein of the Present Invention

The chromoprotein of the present invention is characterized in that itis derived from Cnidopus japonicus, and has the following properties:

-   (1) the absorption maximum wavelength is 610 nm, and fluorescence is    not emitted;-   (2) the molar absorption coefficient is 66,700 at 610 nm; and-   (3) the pH sensitivity of light-absorbing property is stable at    between pH 4 and pH 10.

Cnidopus japonicus is one type of sea anemone belonging to Anthozoa ofCnidaria. Among the types of sea anemone that can be seen in Japan,Cnidopus japonicus has the highest degree of color mutation. Its withersheight is always low, and it has a large number of warts on the bodywall thereof. It has approximately 200 short tentacles. A parent seaanemone discharges developed embryos from its oral part. The dischargedembryos become attached to the body wall of the parent sea anemone.Thereafter, the embryos are further developed, and as a result, theybecome baby sea anemone. Thus, this sea anemone was named KomochiIsoginchaku (a Japanese name meaning “seed sea anemone”). This type ofsea anemone is distributed in the intertidal zones on the rock coastsbetween Hokkaido and Boso Peninsula and also in the zones immediatelybelow the intertidal zones.

It is to be noted that a chromoprotein having the aforementionedproperties was isolated using Cnidopus japonicus as a starting materialin the examples described later, but that the chromoprotein of thepresent invention may also be obtained from forms of sea anemone otherthan Cnidopus japonicus in some cases. Such a chromoprotein is alsoincluded in the scope of the present invention.

As described in the examples below, the chromoprotein of the presentinvention has an absorption maximum wavelength of 610 nm and does notemit fluorescence. In addition, the present chromoprotein has a molarabsorption coefficient of 66,700 at 610 nm. The molar absorptioncoefficient represents the amount of absorbed photons per mole ofmolecule. Since the chromoprotein of the present invention does not emitfluorescence, the chromoprotein of the present invention can be used:(1) as an acceptor molecule (energy receptor) in FRET; (2) indevelopment of a system for converting the energy of applied light intoenergy other than the light; and (3) in introduction of a mutation intothe amino acid sequence of the protein to modify it so that it emitsfluorescence.

In addition, the chromoprotein of the present invention is characterizedin that the pH sensitivity of light-absorbing properties is stable atbetween pH 4 and pH 10. That is to say, in the case of the chromoproteinof the present invention, the peak value of the absorption spectrum doesnot significantly fluctuate in the range between pH 4 and pH 10.Accordingly, even under the same conditions, the chromoprotein of thepresent invention can be used in a broad range of pH environments, andthus, the use of the chromoprotein in vivo has few restrictions.

The examples of the chromoprotein of the present invention include achromoprotein having either one of the following amino acid sequences:

-   (a) the amino acid sequence shown in SEQ ID NO: 1; and-   (b) an amino acid sequence comprising a deletion, substitution    and/or addition of one or several amino acids with respect to the    amino acid sequence shown in SEQ ID NO: 1, and having    light-absorbing properties.

The scope of “one or several” in the phrase “an amino acid sequencecomprising a deletion, substitution and/or addition of one or severalamino acids” is not particularly limited in the present specification.For example, it means 1 to 20, preferably 1 to 10, more preferably 1 to7, further preferably 1 to 5, and particularly preferably 1 to 3.

The term “light-absorbing properties” is used in the presentspecification to mean properties capable of absorbing light having acertain wavelength. For example, an absorption maximum wavelength may be610 nm as in the case of the chromoprotein described in the presentspecification, or the value of the absorption maximum wavelength mayalso be shifted. It is preferable that the pH sensitivity oflight-absorbing properties is stable at between pH 4 and pH 10.

The chromoprotein of the present invention having the amino acidsequence shown in SEQ ID NO: 1 in the sequence listing does not emitfluorescence. In the present invention, one or several amino acids aredeleted, substituted, and/or added with respect to the amino acidsequence shown in SEQ ID NO: 1, so as to produce a protein havingmodified light-absorbing properties, or so as to produce a proteinemitting fluorescence in some cases. The thus produced proteins are alsoincluded in the scope of the present invention.

A specific example of a fluorescent protein produced by such mutation ofamino acids may be a fluorescent protein having an amino acid sequence,wherein, with respect to the amino acid sequence shown in SEQ ID NO: 1,alanine as an amino acid residue at position 28 is substituted byglycine, glutamic acid as an amino acid residue at position 41 issubstituted by methionine, cysteine as an amino acid residue at position145 is substituted by serine, and threonine as an amino acid residue atposition 158 is substituted by isoleucine.

Other specific examples of a fluorescent protein produced by suchmutation of amino acids may include: a chromoprotein having an aminoacid sequence wherein tyrosine as an amino acid residue at position 64is substituted by leucine with respect to the amino acid sequence shownin SEQ ID NO: 1; a chromoprotein having an amino acid sequence whereintyrosine as an amino acid residue at position 64 is substituted bymethionine with respect to the amino acid sequence shown in SEQ ID NO:1; a chromoprotein having an amino acid sequence, wherein glutamic acidas an amino acid residue at position 41 is substituted by leucine, andphenylalanine as an amino acid residue at position 80 is substituted byglycine, with respect to the amino acid sequence shown in SEQ ID NO: 1;a chromoprotein capable of emitting fluorescence, which has an aminoacid sequence wherein tyrosine as an amino acid residue at position 64is substituted by phenylalanine with respect to the amino acid sequenceshown in SEQ ID NO: 1; a chromoprotein capable of emitting fluorescence,which has an amino acid sequence wherein tyrosine as an amino acidresidue at position 64 is substituted by histidine with respect to theamino acid sequence shown in SEQ ID NO: 1; and a chromoprotein capableof emitting fluorescence, which has an amino acid sequence, whereincysteine as an amino acid residue at position 26 is substituted byvaline, cysteine as an amino acid residue at position 143 is substitutedby serine, and proline as an amino acid residue at position 199 issubstituted by leucine, with respect to the amino acid sequence shown inSEQ ID NO: 1.

The method of obtaining the chromoprotein of the present invention isnot particularly limited. The protein may be either a proteinsynthesized by chemosynthesis, or recombinant protein produced by a generecombination technique.

Where a recombinant protein is produced, it is necessary to obtain DNAencoding the protein. Appropriate primers are designed by usinginformation regarding the amino acid sequence shown in SEQ ID NO: 1 ofthe sequence listing of the present specification and the nucleotidesequence shown in SEQ ID NO: 2 thereof. Using these primers, PCR iscarried out by using cDNA library derived from Cnidopus japonicus as atemplate, so that DNA encoding the chromoprotein of the presentinvention can be obtained. The chromoprotein of the present inventioncan be produced by introducing this DNA into an appropriate expressionsystem. Expression in an expression system will be described later inthe present specification.

(2) DNA of the Present Invention

According to the present invention, a gene encoding the chromoprotein ofthe present invention is provided.

Specific examples of DNA encoding the chromoprotein of the presentinvention may include either one of the following DNAs:

-   (a) DNA encoding the amino acid sequence shown in SEQ ID NO: 1; and-   (b) DNA encoding an amino acid sequence, which comprises a deletion,    substitution and/or addition of one or several amino acids with    respect to the amino acid sequence shown in SEQ ID NO: 1, and has    light-absorbing properties.

Other examples of DNA encoding the chromoprotein of the presentinvention may include either one of the following DNAs:

-   (a) the nucleotide sequence shown in SEQ ID NO: 2; and-   (b) a nucleotide sequence comprising a deletion, substitution and/or    addition of one or several nucleotides with respect to the    nucleotide sequence shown in SEQ ID NO: 2, and encoding a protein    having light-absorbing properties.

Further, examples of the DNA having the nucleotide sequence comprising adeletion, substitution and/or addition of one or several nucleotideswith respect to the nucleotide sequence shown in SEQ ID NO: 2, andencoding a protein having light-absorbing properties, may include DNAhaving the nucleotide sequence shown in any one of SEQ ID NOS: 12, 14,16, 18, 20, or 22.

The DNA of the present invention can be synthesized by, for example, thephosphoamidite method, or it can also be produced by polymerase chainreaction (PCR) using specific primers. The DNA of the present inventionis produced by the method described above in the specification.

A method of introducing a desired mutation into a certain nucleic acidsequence is known to a person skilled in the art. For example, knowntechniques such as a site-directed mutagenesis, PCR using degeneratedoligonucleotides, or the exposure of cells containing nucleic acid tomutagens or radioactive rays, are appropriately used, so as to constructDNA having a mutation. Such known techniques are described in, forexample, Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989; and Current Protocolsin Molecular Biology, Supplements 1 to 38, John Wiley & Sons(1987-1997).

(3) Recombinant Vector of the Present Invention

The DNA of the present invention can be inserted into a suitable vectorand used. The type of a vector used in the present invention is notparticularly limited. For example, it may be either a vector that canautonomously replicate (e.g., a plasmid, etc.), or vector that isincorporated into the genomes of host cells when it is introduced intothe host cells and is then replicated together with the chromosome intowhich it is incorporated.

The vector used in the present invention is preferably an expressionvector. In an expression vector, elements necessary for transcription(e.g., a promoter, etc.) are functionally ligated to the DNA of thepresent invention. The promoter is a DNA sequence which shows atranscriptional activity in host cells, and it is appropriately selecteddepending on the type of host cells.

Examples of a promoter which can operate in bacterial cells may includea Bacillus stearothermophilus maltogenic amylase gene promoter, aBacillus licheniformis alpha-amylase gene promoter, a Bacillusamyloliquefaciens BAN amylase gene promoter, a Bacillus subtilisalkaline protease gene promoter, a Bacillus pumilus xylosidase genepromoter, P_(R) and P_(L) promoters of phage rhamda, and lac, trp andtac promoters of Escherichia coli.

Examples of a promoter which can operate in mammalian cells may includean SV40 promoter, an MT-1 (metallothionein gene) promoter, and anadenovirus-2 major late promoter. Examples of a promoter which canoperate in insect cells may include a polyhedrin promoter, a P10promoter, an Autographa californica polyhedrosis basic protein promoter,a baculovirus immediate-early gene 1 promoter, and a baculovirus 39Kdelayed-early gene promoter. Examples of a promoter which can be operatein yeast host cells may include promoters derived from yeast glycolyticgenes, an alcohol dehydrogenase gene promoter, a TPI1 promoter, and anADH2-4c promoter.

Examples of a promoter which can operate in filamentous cells mayinclude an ADH3 promoter and a tpiA promoter.

In addition, an appropriate terminator such as a human growth hormoneterminator, or a TPI1 terminator or ADH3 terminator for fungal cells,may be functionally bound to the DNA of the present invention, asnecessary. The recombinant vector of the present invention may furtherhave elements such as a polyadenylation signal (e.g., one derived fromSV40 or the adenovirus 5E1b region), a transcription enhancer sequence(e.g., an SV40 enhancer), or a translation enhancer sequence (e.g., oneencoding the adenovirus VA RNA).

The recombinant vector of the present invention may further comprise aDNA sequence which enables the replication of the recombinant vector inhost cells. SV40 replication origin is an example of such a sequence(when the host cells are mammalian cells).

The recombinant vector of the present invention may further comprise aselective marker. Examples of such a selective marker may include genes,complements of which are absent from host cells, such as a dihydrofolatereductase (DHFR) gene or a Shizosaccharomyces pombe TPI gene, and drugresistant genes such as ampicillin, kanamycin, tetracycline,chloramphenicol, neomycin or hygromycin-resistant genes.

A method for ligating the DNA of the present invention, a promoter and,as desired, a terminator and/or a secretory signal sequence to oneanother and inserting these items into a suitable vector is known to aperson skilled in the art.

(4) Transformant of the Present Invention

A transformant can be produced by introducing the DNA or recombinantvector of the present invention into a suitable host.

Any cell can be used as a host cell into which the DNA or recombinantvector of the present invention is introduced, as long as the DNAconstruct of the present invention can be expressed therein. Examples ofsuch a cell may include bacteria, yeasts, fungal cells, and highereukaryotic cells.

Examples of bacteria may include Gram-positive bacteria such as Bacillusor Streptomyces, and Gram-negative bacteria such as Escherichia coli.These bacteria may be transformed by the protoplast method or otherknown methods, using competent cells.

Examples of mammalian cells may include HEK 293 cells, HeLa cells, COScells, BHK cells, CHL cells, and CHO cells. A method of transformingmammalian cells and expressing the introduced DNA sequence in the cellsis also known. Examples of such a method may include theelectroporation, the calcium phosphate method, and the lipofectionmethod.

Examples of yeast cells may include those belonging to Saccharomyces orShizosaccharomyces. Examples of such cells may include Saccharomycescerevisiae and Saccharomyces kluyveri. Examples of a method ofintroducing a recombinant vector into yeast host cells may include theelectroporation, the spheroplast method, and the lithium acetate method.

Examples of other fungal cells may include those belonging toFilamentous fungi such as Aspergillus, Neurospora, Fusarium orTrichoderma. Where Filamentous fungi are used as host cells,transformation can be carried out by incorporating DNA constructs intohost chromosomes, so as to obtain recombinant host cells. Incorporationof DNA constructs into the host chromosomes is carried out by knownmethods, and such known methods may include homologous recombination andheterologous recombination.

Where insect cells are used as host cells, both a vector into which arecombinant gene is introduced and a baculovirus are co-introduced intoinsect cells, and a recombinant virus is obtained in the culturesupernatant of the insect cells. Thereafter, insect cells are infectedwith the recombinant virus, so as to allow the cells to express proteins(described in, for example, Baculovirus Expression Vectors, A LaboratoryManual; and Current Protocols in Molecular Biology, Bio/Technology, 6,47 (1988)).

The Autographa californica nuclear polyhedrosis virus, which is a virusinfecting to insects belonging to Barathra brassicae, can be used asbaculovirus.

Examples of insect cells used herein may include Sf9 and Sf21, which areSpodoptera frugiperda ovarian cells [Baculovirus Expression Vectors, ALaboratory Manual, W. H. Freeman & Company, New York, (1992)], andHiFive (manufactured by Invitrogen), which are Trichoplusia ni ovariancells.

Examples of the method of co-introducing both a vector into which arecombinant gene has been introduced and the above baculovirus intoinsect cells to prepare a recombinant virus may include the calciumphosphate method and the lipofection method.

The above transformant is cultured in an appropriate nutritive mediumunder conditions enabling the introduced DNA construct to be expressed.In order to isolate and purify the protein of the present invention fromthe culture product of the transformant, common methods of isolating andpurifying proteins may be used.

For example, where the protein of the present invention is expressed ina state dissolved in cells, after completion of the culture, cells arerecovered by centrifugal separation, and the recovered cells aresuspended in a water type buffer. Thereafter, the cells aredisintegrated using an ultrasonic disintegrator or the like, so as toobtain a cell-free extract. A supernatant is obtained by centrifugingthe cell-free extract, and then, a purified sample can be obtained fromthe supernatant by applying, singly or in combination, the followingordinary protein isolation and purification methods: the solventextraction, the salting-out method using ammonium sulfate or the like,the desalting method, the precipitation method using an organic solvent,the anion exchange chromatography using resins such as diethylaminoethyl(DEAE) sepharose, the cation exchange chromatography using resins suchas S-Sepharose FF (manufactured by Pharmacia), the hydrophobicchromatography using resins such as butyl sepharose or phenyl sepharose,the gel filtration method using a molecular sieve, the affinitychromatography, the chromatofocusing method, and the electrophoresissuch as isoelectric focusing.

(5) Use of the Chromoprotein of the Present Invention and a FusionProtein Comprising the Same

The chromoprotein of the present invention can be fused with anotherprotein, so as to construct a fusion protein. The type of said anotherprotein to be fused to the chromoprotein of the present invention is notparticularly limited, and preferred examples may include a protein whichinteracts with another molecule. The examples may include a receptorprotein or ligand thereof, antigen, antibody and the like.

A method of obtaining the fusion protein of the present invention is notparticularly limited. It may be either a protein synthesized bychemosynthesis, or recombinant protein produced by a gene recombinationtechnique.

Where a recombinant fusion protein is produced, it is necessary toobtain DNA encoding the protein. The DNA encoding the chromoprotein ofthe present invention and the DNA encoding the another protein to befused to the chromoprotein, can be obtained by the method as mentionedabove in this specification or by the method similar to it. Then, theseDNA fragments are ligated to one another by a gene recombinationtechnique, so that DNA encoding the desired fusion protein can beobtained. This DNA is then introduced into an appropriate expressionsystem, so that the fusion protein of the present invention can beproduced.

FRET (fluorescence resonance energy transfer) has been known as a meansfor analyzing the interaction between molecules. In FRET, for example, afirst molecule labeled with a cyan fluorescent protein (CFP) acting as afirst fluorescent protein is allowed to coexist with a second moleculelabeled with a yellow fluorescent protein (YFP) acting as a secondfluorescent protein, so as to allow the yellow fluorescent protein (YFP)to act as an acceptor molecule and to allow the cyan fluorescent protein(CFP) to act as a donor molecule. Thus, FRET (fluorescence resonanceenergy transfer) is allowed to take place between both molecules, so asto visualize the interaction between the first and second molecules.Namely, in FRET, different dyes are introduced into two types ofmolecules. One dyes with a higher energy level (a donor molecule) isselectively excited, and the fluorescence of the dye is measured.Long-wavelength fluorescence from the other dye (an acceptor molecule)is also measured. The interaction between the molecules is visualized byusing the difference between the amounts of both fluorescences. Onlywhen both dyes are adjacent to each other due to the interaction of thetwo types of molecules, a decrease in the fluorescence of the donormolecule and an increase in the fluorescence of the acceptor moleculeare observed by single wavelength excitation dual wavelength photometry.However, in a case where a chromoprotein is used as an acceptormolecule, a decrease in the fluorescence of the donor molecule occursonly when both dyes are adjacent to each other by the interaction of thetwo types of molecules. Such a decrease can be observed by singlewavelength excitation single wavelength photometry. Thus, the use of thechromoprotein of the present invention enables facilitation ofmeasurement apparatuses.

The chromoprotein of the present invention is particularly advantageouswhen it is used as an acceptor molecule in FRET (fluorescence resonanceenergy transfer). That is to say, a fused form (a first fused form) ofthe chromoprotein of the present invention and a test substance is firstproduced. Then, a fused form (a second fused form) of another testsubstance interacting with the above test substance and anotherfluorescent protein is produced. Thereafter, the first fused form isallowed to interact with the second fused form, and the generatedfluorescence is analyzed, so that the interaction between theaforementioned two types of test substances can be analyzed. FRET(fluorescence resonance energy transfer) using the chromoprotein of thepresent invention may be carried out either in a test tube or in a cell.

(6) Kit of the Present Invention

The present invention provides a light-absorbing reagent kit comprisingat least one which is selected from the chromoprotein, fusion protein,DNA, recombinant vector or transformant, which are described in thepresent specification. The kit of the present invention can be producedfrom commonly used materials that are known per se, by using commonmethods.

Reagents such as the chromoprotein or the DNA are dissolved in anappropriate solvent, so that the reagents can be prepared in a formsuitable for conservation. Water, ethanol, various types of buffersolution, etc. can be used as such a solvent.

The present invention will be further described in the followingexamples. However, the present invention is not limited by theseexamples.

EXAMPLES Example 1 Isolation of Gene Encoding Novel Chromoprotein fromSea Anemone

(1) Extraction of Total RNA

A chromoprotein gene was isolated from sea anemone emitting a greencolor. Cnidopus japonicus emitting a green color was used as a material.Frozen Cnidopus japonicus was crushed in a mortar. 7.5 ml of “TRIzol”(GIBCO BRL) was added to 1 g (wet weight) of Cnidopus japonicus, and themixture was homogenized, followed by centrifugation at 1,500×g for 10minutes. 1.5 ml of chloroform was added to the supernatant. The mixturewas stirred for 15 seconds and then left at rest for 3 minutes. Theresultant product was centrifuged at 7,500×g for 15 minutes. 3.75 ml ofisopropanol was added to the supernatant. The mixture was stirred for 15seconds and then left at rest for 10 minutes. The resultant product wascentrifuged at 17,000×g for 10 minutes. The supernatant was discarded,and 6 ml of 70% ethanol was added thereto. The obtained mixture wascentrifuged at 17,000×g for 10 minutes. The supernatant was discarded,and the precipitate was dissolved in 200 μl of DEPC water. Total RNAdissolved in the DEPC water was 100 times diluted, and the values ofO.D.260 and O.D.280 were measured, so as to determine the concentrationof RNA. 1.2 mg of the total RNA was obtained from a green individual.

(2) Synthesis of First Stand cDNA

cDNA (33 μl) was synthesized from 4 μg of the total RNA using a kit forsynthesizing first strand cDNA, “Ready To Go” (Amersham Pharmacia).

(3) Degenerated PCR

Using 3 μl out of the synthesized first strand cDNA (33 μl) as atemplate, PCR was carried out. Primers were designed and produced bycomparing the amino acid sequences of known fluorescent proteins,extracting similar portions, and converting them into nucleotidesequences. The sequences of the used primers are shown below:

5′-GGNGSNCCNHTNSCNTT-3′; (primer 1) (SEQ ID NO: 3) and5′-AACTGGAAGAATTCGCGGCCGCAGAATTTTTTTTTTTTTTTTTT-3′, (primer 2) (SEQ IDNO: 4)wherein N represents inosine, S represents C or G, and H represents A,T, or C.

Composition of PCR reaction solution Template (first strand cDNA) 3 μl X10 taq buffer 5 μl 2.5 mM dNTPs 4 μl 100 uM primer 3 1 μl 100 uM primer4 1 μl Milli Q 35 μl  Taq polymerase (5 U/μl) 1 μl

-   RCR Reaction Conditions-   94° C., 1 minute (PAD)-   94° C., 30 seconds (denaturation)-   52° C., 30 seconds (annealing of the primers to the template)-   72° C., 1 minute (elongation of the primers)

30 cycles consisting of the above 3 steps were carried out. Theannealing temperature was decreased 0.3° C. per cycle. That is to say,the annealing temperature in the 30^(th) cycle was 43° C.

-   72° C., 7 minutes (final elongation)-   Retention at 4° C.

Using 1 μl of an amplified product obtained as a result of the first PCRreaction as a template, PCR was carried out once again under the sameconditions. A 800-bp fragment (derived from the green individual) wascut out by agarose gel electrophoresis and then purified. This 800-bpfragment contained a 3′-UTR portion as a whole.

(4) Subcloning and Sequencing

The purified DNA fragment was ligated to a pT7-blue vector (Novagen).Escherichia coli (TG1) was transformed with the vector, and the obtainedtransformants were subjected to blue white selection. Thereafter,plasmid DNA was purified from white colonies of Escherichia coli. Thenucleotide sequence of the inserted DNA fragment was determined by a DNAsequencer. The obtained nucleotide sequence was compared with thenucleotide sequences of other fluorescent protein genes to confirm thatthe nucleotide sequence of the DNA was derived from a fluorescentprotein. 5′-RACE and 3′-RACE methods were applied to a gene that hadbeen confirmed to be a part of a fluorescent protein gene, so as tocarry out the cloning of a full-length gene.

(5) 5′-RACE Method

In order to determine the nucleotide sequence of the 5′-terminal side ofthe DNA fragment obtained by degenerated PCR, the 5′-RACE method wasapplied using 5′-RACE System for Rapid Amplification of cDNA Ends,Version 2.0 (GIBCO BRL). 3 μg of the total RNA prepared in (1) above wasused as a template.

For the first amplification of DC-tailed cDNA of the green individual,the following primers were used:

(SEQ ID NO: 5) 5′-GGCCACGCGTCGACTAGTACGGGNNGGGNNGGGNNG-3′; (primer 3)and (SEQ ID NO: 6) 5′-AGACGAGGCAATTTCCATCAAG-3′, (primer 4)wherein N represents inosine.

For the second amplification, the following primers were used:

(SEQ ID NO: 7) 5′-GGCCACGCGTCGACTAGTAC-3′; (primer 5) and (SEQ ID NO: 8)5′-GGCTACGCTTCCATATTGGCAGTT-3′. (primer 6)PCR reaction conditions and the like were determined in accordance withthe protocols attached with the kit.

The 350-bp amplified band was cut out by agarose gel electrophoresis andthen purified. The purified DNA fragment was ligated to a pT7-bluevector (Novagen). Escherichia coli (TG1) was transformed with thevector, and the obtained transformants were subjected to blue whiteselection. Thereafter, plasmid DNA was purified from white colonies ofEscherichia coli. The nucleotide sequence of the inserted DNA fragmentwas determined by a DNA sequencer. The entire nucleotide sequence isshown in SEQ ID NO: 2, and the entire amino acid sequence is shown inSEQ ID NO: 1.

Example 2 Expression of Protein in Escherichia coli

Primers corresponding to the N- and C-termini of the protein wereproduced from the obtained full-length nucleotide sequence. PCR wascarried out using the primers and the first strand cDNA prepared in (2)above as a template. The used primers are as follows:

(SEQ ID NO: 9) 5′-CGGGATCCGACCATGGCTTCCAAAATCAGC-3′; (primer 7) and (SEQID NO: 10) 5′-CCGGAATTCTTAATTGTGACCAAGTTTAGATGGGCA-3′. (primer 8)

Composition of PCR reaction solution Template (first strand cDNA) 3 μl X10 pyrobest buffer 5 μl 2.5 mM dNTPs 4 μl 100 μM primer 7 1 μl 100 μMprimer 8 1 μl Milli Q 35 μl  Pyrobest polymerase (5 U/μl) 1 μl

-   RCR Reaction Conditions-   94° C., 1 minute (PAD)-   94° C., 30 seconds (denaturation)-   55° C., 30 seconds (annealing of the primers to the template)-   72° C., 1 minute (elongation of the primers)

30 cycles consisting of the above 3 steps were carried out.

-   72° C., 7 minutes (final elongation)-   Retention at 4° C.

An amplified band of approximately 700 bp was cut out by agarose gelelectrophoresis and then purified. The purified DNA fragment wassubcloned into the BamHI-EcoRI site of a pRSET vector (Invitrogen), andit was then allowed to express in Escherichia coli (JM109-DE3). Sincethe expressed protein was constructed such that His-tag was attached tothe N-terminus thereof, it was purified with Ni-Agarose gel (QIAGEN).Purification was carried out in accordance with the attached protocols.

Example 3 Analysis of Protein

(1) Analysis of Light-absorbing Properties

The light-absorbing properties of the protein expressed in Example 2were analyzed.

An absorption spectrum was measured using a 50 mM HEPES solution (pH7.5) containing a 20 μM chromoprotein. A molar absorption coefficientwas calculated from the peak value of this spectrum. In the case of thechromoprotein derived from the green individual (referred to as KGr),the absorption peak was observed at 610 nm, and no fluorescence wasdetected (Table 1, FIG. 1).

TABLE 1 Properties of chromoprotein (KGr) Fluores- Molar pH Absorptioncence absorption quantum sensi- Number of maximum maximum coefficientyield tivity amino acids 610 nm — 66,700 — Non 232 (610 nm)(1) Measurement of pH Sensitivity

The pH sensitivity of the protein expressed in Example 2 was analyzed.

The absorption spectrum of the protein was measured in the following 100mM buffer solution (FIG. 2).

The following buffer solutions were used for each pH:

-   pH 4 and 5: Acetate buffer-   pH 6: MES buffer-   pH 7 and 8: HEPES buffer-   pH 9 and 10: Glycine buffer

The peak value did not significantly change at any pH.

Example 4 Modification of KGr

In KGr, A at position 28 was substituted by G, E at position 41 wassubstituted by M, C at position 145 was substituted by S, and T atposition 158 was substituted by I, so that the KGr was modified to havean absorption peak at 444 nm and to emit yellow fluorescence with a peakat 534 nm (FIG. 3).

Example 5 Modification of Properties of KGr by Amino Acid Substitution

Y at position 64 that is a chromophore-forming amino acid (QYG) of KGrwas substituted by L or M, so that the absorption peak became 418 nm andso that the absorption peak was shifted from the original absorptionpeak at 610 nm to the shorter wavelength side (FIGS. 1 and 2). The aminoacid sequence of the protein wherein Y at position 64 was substituted byL is shown in SEQ ID NO: 11, and the nucleotide sequence thereof isshown in SEQ ID NO: 12. The amino acid sequence of the protein wherein Yat position 64 was substituted by M is shown in SEQ ID NO: 13, and thenucleotide sequence thereof is shown in SEQ ID NO: 14.

E at position 41 was substituted by L, and F at position 80 wassubstituted by G, so that the absorption peak become 528 nm and so thatthe absorption peak was shifted from the original absorption peak at 610nm to the shorter wavelength side (FIG. 3). The amino acid sequence ofthis protein is shown in SEQ ID NO: 15, and the nucleotide sequencethereof is shown in SEQ ID NO: 16.

Y at position 64 that is a chromophore-forming amino acid (QYG) wassubstituted by F, so that the absorption peak became 412 nm, and so thatthe absorption peak was shifted from the original absorption peak at 610nm to the shorter wavelength side and the protein was further modifiedto emit fluorescence with a peak at 504 nm (FIGS. 4 and 5). The aminoacid sequence of this protein is shown in SEQ ID NO: 17, and thenucleotide sequence thereof is shown in SEQ ID NO: 18.

Y at position 64 that is a chromophore-forming amino acid (QYG) wassubstituted by H, so that the absorption peak became 418 nm, and so thatthe absorption peak was shifted from the original absorption peak at 610nm to the shorter wavelength side and the protein was further modifiedto emit fluorescence with a peak at 520 nm (FIGS. 6 and 7). The aminoacid sequence of this protein is shown in SEQ ID NO: 19, and thenucleotide sequence thereof is shown in SEQ ID NO: 20.

C at position 26 was substituted by V, C at position 143 was substitutedby S, and P at position 199 was substituted by L, so that the absorptionpeak became 585 nm, and so that the absorption peak was shifted from theoriginal absorption peak at 610 nm to the shorter wavelength side andthe protein was further modified to emit fluorescence with a peak at 625nm. This fluorescent protein was defined as KGr Rb (FIGS. 8 and 9). Theamino acid sequence of this protein is shown in SEQ ID NO: 21, and thenucleotide sequence thereof is shown in SEQ ID NO: 22.

INDUSTRIAL APPLICABILITY

The present invention provides a novel chromoprotein derived fromCnidopus japonicus. The chromoprotein of the present invention hasdesired fluorescence properties and low pH sensitivity. Thus, it isuseful for molecular biology analysis.

1. An isolated DNA of either one of the following: (a) DNA encoding theamino acid sequence shown in SEQ ID NO: 1, wherein said SEQ ID NO: 1 haslight absorbing properties but does not emit fluorescence, or (b) DNAencoding the amino acid sequence shown in SEQ ID NO: 1, which furthercomprises a deletion, substitution and/or addition of one to ten aminoacids and has modified light-absorbing properties and/or emitsfluorescence.
 2. An isolated DNA having the nucleotide sequence shown inSEQ ID NO:
 2. 3. An isolated DNA having the nucleotide sequence shown inany one of SEQ ID NOS: 12, 14, 16, 18, 20, or
 22. 4. A recombinantvector having the DNA of claim
 1. 5. An isolated transformant comprisingthe DNA of claim 1 or a recombinant vector having the DNA of claim
 1. 6.A light-absorbing reagent kit comprising the isolated DNA of claim
 1. 7.A light-absorbing reagent kit comprising the recombinant vector of claim4.
 8. A light-absorbing reagent kit comprising the isolated transformantof claim 5.